Recombinant adenoviral vectors and their utility in the treatment of various types of fibrosis: hepatic, renal, pulmonary, as well as hypertrophic scars

ABSTRACT

The use of gene therapy for the treatment of different kinds of fibrosis in human beings is disclosed. The purpose is the use of “therapeutic2 genes specifically directed to target organs to revert and/or prevent the development of the fibrosis process.  
     The potential application of gene therapy to patients with fibrosis and/or cirrhosis will depend to a large extent on the successful delivery of genes which encode for therapeutic proteins to livers with severe fibrosis and that these genes which encode for proteins human MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-13; human uPA wild type and/or modified (or its truncated version), the truncated receptor for TGF-β type II and Smad-7 can be directed by adenovirus and/or other recombinant vectors that cannot transduce (infect) others organs. The recombinant adenoviruses (AdR) are vectors highly efficient for the transduction of therapeutic genes to diverse target cells. We have proved that they can carry genes to cirrhotic livers.  
     The delivery of therapeutic genes through such adenoviral vectors and other recombinant vectors could also be performed using cationic and anionic liposomes (DOTMA).  
     Therefore, we propose the use of this patent to be applied in the same manner to:  
     Renal fibrosis  
     Pulmonary fibrosis  
     Hypertrophic and keloid scars (skin fibrosis), and  
     Other kinds of fibrosis.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to the creation of RECOMBINANTADENOVIRAL vectors bearing exogenous genes that encode for therapeuticproteins useful in the treatment of HEPATIC cirrhosis and generalizedFIBROSIS, such as renal FIBROSIS, pulmonary FIBROSIS, HYPERTROPHIC scarsand keloid of the skin, and/or in other target organs susceptible tosuffer from it. It also relates to a mechanism of tissue-specificrecognition of the affected cells by means of delivery of therapeuticgenes to cirrhotic organs.

[0002] Moreover, the invention provides an effective way for thetreatment of fibrosis through the employment of recombinant adenoviralvectors which are claimed here, as well as the process to prepare thesevectors, the pharmaceutical composition that contains them, and theirtherapeutic uses in the treatment of several fibrosis, which has greatcommercial expectancy in the pharmaceutical industry and also presentsan important alternative as gene therapy for the treatment ofchronic-degenerative diseases characterized by fibrosis, with greattherapeutic application in the field of Medicine.

INTRODUCTION Physiopathology of Hepatic Cirrhosis

[0003] Hepatic cirrhosis is a disease resulting from hepatic chronicdamage. Damage might be toxic (chronic ingestion of alcohol), infectious(viral hepatitis, mainly by hepatitis B and/or C virus), immunological,(primary biliary cirrhosis), by biliary obstruction, (secondary biliarycirrhosis), metabolic (Wilson's disease). All forms of cirrhosis havecharacteristics in common: synthesis and excessive deposition ofproteins of extracellular matrix (ECM), mainly collagen I and to alesser extent collagens IV and II), and consequently the formation ofnodules of hepatocytes, abnormal vascularization and portal hypertension(Antoni P P, Ishak K G, Nayak Nc, Poulsen H E, Sheuer P J, Sobin L H.These physiopathological processes lead to an alteration in the bloodsupply and in consequence in the nutrition of hepatic cells. Regardlessof the ethiological agent and morphologic differences, all forms ofcirrhosis have as a common end, hepatic failure causing the patient'sdeath.

[0004] As a consequence of the excessive deposition of collagen proteinsin the sub-endothelial space of the sinusoids (Space of Disse), variouschanges occur in the hepatic microenvironment: loss of hepatocyte villi,formation of a basement membrane composed by collagens IV and I coveringthe sinusoids, and loss of the fenestration of endothelial cells whichforms the sinusoids. All this process is known as “capillarization” ofthe sinusoids. (Scott L. Friedman The cellular basis of hepaticfibrosis: Mechanisms and treatment strategies. The New England Journalof Medicine 1993, vol. 328 No. 25:1828-1835). Thus, the liver is notable to maintain the physiologic concentration of solutes in theterminal hepatic vein, in other words, HEPATIC failure sets in. Thiscapillarization, with the formation of the continuous endothelia(collagen of basement membrane) and the accumulation of other collagenicproteins, represents a barrier to the normal and bidirectional exchangeof molecules between the plasma and hepatocytes, as can be appreciatedin FIG. 1, where hepatic cirrhosis is characterized by the accumulationin the liver of type I collagen. With an excessive deposition of thisprotein, the free exchange of nutrients between blood and liver cells isimpeded, the inactivation of toxic agents by this organ can not becarried out, becoming this the main cause of the pathophysiology of thedisease. To date, no therapeutic agent that could revert and/or preventwith a 100% effectiveness the progressive accumulation of hepaticcollagen has been described.

[0005] Such physiopathological alterations presented in hepaticcirrhosis are constant and common for the organs that also undergofibrosis, such as, lung, heart, kidney, skin, among others, which shouldbe not considered as limitations of the scope of protection of thisinvention. Therefore, the methodology presented here for the treatmentof hepatic cirrhosis could be applied also to those organs that aresusceptible to, or are affected by fibrosis.

Viral Vectors and Hepatic Gene Therapy

[0006] This technology can be implemented with viral or non-viralvectors. Previous studies have been designed using plasmids andliposomes (DOTMA), cationic and anionic, etc. Among the methodsemploying viral vectors, the most commonly used include the use ofretrovirus and adenovirus.

[0007] In a number of protocols, retroviral vectors have been used tointroduce genes in hepatocytes (JT, and Curiel DT, Adenoviruses asVectors for gene Therapy. Science and Medicine/1997 44-53). However,precautions have to be taken since these vectors can generate potentialreplication-competent viruses. Among the advantages of these vectors istheir ability to integrate their genome in a stable way in thechromosomes of the guest cell, which confers the possibility ofexpression, in an indefinite way, of the therapeutic transgene cloned inthe retrovirus. On the other hand, up to date, no study has reportedincidences of mutagenesis by insertion or activation of oncogenes by theincorporation of the replication-deficient retrovirus. Nevertheless, theuse of retroviral vectors to transduce genes to the liver is limited forthe following considerations: 1) these vectors infect only cells whichactively divide and 2) very low viral particles titers are obtained inthe packing cell lines used to amplify these viruses (Graham F L, andVan Der Eb A J. A New Technique for the Assay of Infectivity of HumanAdenovirus 5 DNA. Virology 1973, 52:456-467). These two limitations havebeen successfully overcome in other Gene Therapy protocols through theinduction of hepatocytes proliferation “in vivo”, through the useHepatic Growth Factors and through partial hepatectomy, surgicalprocedure by which the removal of 70% of liver mass induces division ofthe remaining hepatic cells “in vivo”. The use of Lentiviral vectors haspermitted to overcome partially said limitations, because they are ableto transduce cells which are not actually dividing.

BACKGROUND OF THE INVENTION

[0008] Hepatic cirrhosis is a chronic illness of the liver, wherediffuse cell necrosis and a limited regeneration of parenchymal hepaticcells result in diffuse percentage increase of connective tissue,causing the distortion of lobular hepatic architecture and inducinghemodynamic alterations. Therefore, some strategies for the treatment ofhepatic cirrhosis could include the prevention and/or reversion of the“fibrogenic process”, stimulation of hepatic mitosis and re-arrangementof the architecture of hepatic tissue. The documents of the state of theart related to the present invention are mentioned hereinafter only asreferences.

[0009] U.S. Pat. No. 5,240,846 refers to the use of gene therapy called“CFTR”, which induces a stable correction of the regulation of thechlorine channel. This defect is present in epithelial cells. In saidinvention, adenoviral recombinant vectors are used as well as plasmidicvectors. However, it does not have any association with the therapeuticsgenes of the present invention. Likewise, U.S. Pat. No. 5,910,487,describes the use of plasmidic vectors for sending therapeuticmolecules, but there is no association with the delivery of genes ofmetalloproteases MMP-8 latent and/or active, MMP-1, MMP-2, MMP-9,MMP-13; or “uPA (wild type uPA and/or its modified versions) or “Smad7”or the truncated receptors for transforming growth factor-β (TGF-β typeII) as presented here. U.S. Pat. No. 5,827,703 refers to the use ofadenoviral vector and modified adenoviral vector to send genes, howevernone of these vectors contain the genes used in the present inventionfor the treatment of fibrosis.

[0010] U.S. Pat. No. 5,770,442 claims the use of a recombinantadenovirus that contains one gene directing the expression of a proteincalled “fiber” or a protein called “Fiber-chimera”, however said patentdoes not specifically mention, which one is the therapeutic gene. Also,a method of gene therapy involving the use of such adenovirus and avector of transference for the generation of such recombinant adenovirusis presented. However, nothing is mentioned with regard to the use oftherapeutic genes cloned and inserted in recombinant adenoviral vectorsused in this invention in fibrotic livers, or to other target organssuch as kidney, lung, and hypertrophic scars and others. Thesetherapeutic genes are the gene that codes for human metalloproteasesMMP-8, latent and/or active, MMP-1, MMP-2, MMP-9 and MMP-13; humanurokinase Plasminogen Activator (wild type and/or modified huPA), Smad7,and the truncated receptor for TGF-β type II, claimed herein. Othermembers of the family of genes represented are also included.

[0011] U.S. Pat. No. 5,166,320 refers to the use of a targeted deliverysystem to introduce exogenous genes in mammalian hepatic cells. Butthere is no association with putative genes directly sent to cirrhoticlivers or to fibrotic kidney or lungs.

[0012] U.S. Pat. No. 5,872,154, describes a method to reduce the immuneresponse induced by an adenoviral recombinant vector and a selectedimmune modulator, which functions by inhibiting the formation ofneutralizing antibodies and/or reducing the death of the virallyinfected cells.

[0013] U.S. Pat. No. 5,871,982, is directed to a hybrid vector, in whicha portion of an adenovirus is included, together with a portion of anadeno-associated viral vector that contains a selected transgene. Ahybrid virus consisting of the union of a conjugate with a polycation toa gene mesh of the adeno-associated viral vector to form a simpleparticle is also described. This is contrary to the present invention inwhich no hybrid viruses are employed, only adenoviral vectors. Besides,in the above-mentioned patent the gene, transgene or therapeutic geneused is not stated.

[0014] U.S. Pat. No. 5,856,152 is directed to the creation of a hybridvector which contains the portion of an adenoviral vector in combinationwith an adeno-associated virus and a selected gene. Thorough it largequantities of recombinant vectors are produced, but they are notcarrying cloned therapeutic genes as is described in this invention, inwhich specific therapeutic genes for the treatment of renal and hepaticfibrosis and hypertrophic scars are used.

[0015] U.S. Pat. No. 5,547,932 claims a compound of complexes of nucleicacids for transfecting eucaryotic cells. These complexes are formed bynucleic acids and another substance with affinity for nucleic acids andoptionally an internalizing factor, such as a virus or a component ofthe virus that can be conjugated. It also uses components of specificadenoviral vectors or specific viruses such as Ad2 or Ad5, but does notmention the genes that are internalized in the cell cytoplasm andeventually in the nucleus of these eucaryotic cells. Similarly, U.S.Pat. No. 5,521,291, is related to conjugated adenovirus bound through anantibody to a substance with affinity to nucleic acids. In this wayrecombinant genes are transported to the interior of eucaryotic cells.These conjugated complexes and nucleic acids are internalized in thecell, but the genes that can be sent are not specifically mentioned. Insaid patent, contrary to what is described in the instant invention, theuse of such adenovirus to treat fibrosis or hepatic cirrhosis or anyanother type of fibrosis is not mentioned.

[0016] U.S. Pat. No. 5,585,362, relates to an improved adenoviral vectorand methods to obtain and use such vectors. The use of adenoviralvectors is not mentioned in said patent. However the adenoviral vectorsdescribed in the present invention were used like vectors for sendingtherapeutic genes.

[0017] U.S. Pat. No. 5,756,086, claims an adenovirus, which isrepresented by a protein called “fiber”, the adenovirus also includes aligand, that is specific for a receptor located in a specific cell type.This adenovirus can have at least a portion of this protein called“fiber” and it can be removed and replaced with a ligand, which isspecific for a receptor in specific cells of the economy, such ashepatocytes. This adenovirus can include a gene that codes for atherapeutic agent. Based on the previous statement, the outstandingtechnical difference of the instant invention compared to the state ofthe art, is the specificity of the therapeutic agent as humanmetalloproteases MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and.MMP-13; human uPA (urokinase Plasminogen Activator, wild type and/ormodified), the truncated receptor for TGF-β type II and “Smad7” for thetreatment of various fibrosis.

[0018] U.S. Pat. No. 5,895,759 claims a tissue-specific vector (liver)for gene therapy that can be used to send genes to a damaged liver.These vectors are chemically or enzyme coupled to a promoter and canalso be coupled to an antibody packaged in a polypeptidic envelope.Besides, the vector or the virus to be assayed is the hepatitis B virus.Thus the sending of genes to damaged livers described in this patentmakes use of a system completely different from the one of thisinvention, and there is no relation with the process of fibrosis orcirrhosis to be treated.

[0019] U.S. Pat. No. 5,559,099 describes an adenoviral recombinantvector that contains a chimeric protein from the adenovirus calledpentona, which includes a non-pentona sequence and a therapeutic gene todevelop a gene therapy method involving the use of such adenovirus,transference adenoviral vectors for the recombination of such adenoviralvectors containing a therapeutic gene.

[0020] U.S. Pat. No. 5,885,808 claims also the use of adenovirus withbonding molecules of adenovirus to different cells, the molecules ofwhich have been modified, as in U.S. Pat. Nos. 5,846,782 and 5,712,136,in which adenoviral vectors are employed, which have been modified tocontain different peptidic domains.

[0021] Finally, U.S. Pat. No. 5,670,488 relates to vectors for genetherapy, which are especially useful for cystic fibrosis and alsomentions the development of methods for the use of these vectors. Thepossible relation of the instant invention to the mentioned state of theart refers to the use of adenoviral vectors, that can be modified, aswell as the use of inducible promoters driving the expression of genesto be inserted in these adenoviral vectors. However, the technicalcharacteristics of the present invention are focused on the specific useof therapeutic genes to treat fibrosis of different kinds: hepatic,renal and pulmonary fibrosis, as well as hypertrophic scars.

[0022] The importance of the present invention, contrary to the state ofthe art described in the above-mentioned documents, is based on thetechnical characteristics of the invention itself, as well as on theadditional advantages derived from the same, which are described withmore details below.

Adenoviral Vectors

[0023] In the instant invention, the use of adenoviral vectors wasdetermined based on several considerations: 1) these vectors can begenerated to very high titers of infectious particles per ml.:(10⁹-10¹⁰); 2) they infect a great variety of cells, however, when theyare administered i.v., most of them are located in the hepatic organ; 3)they transfer efficiently genes to cells that are not dividing, and 4)they are seldom integrated in the guest genome, which avoids the risk ofcellular transformation by insertional mutagenesis (Douglas J T, andCuriel D T. Adenoviruses as Vectors for gene Therapy. Science andmedicine, March/April 1997. 44-53 and Zern A M, and Kresina T F. HepaticDrug delivery and Gene Therapy. Hepatology 1997, Vol. 25, No. 2,484-491).

[0024] Adenovirus are probably the most promising vehicles or vectorsfor the delivery of genes in the protocols of gene therapy in humanbeings, since they possess a unique attribute that provides them greatstability when they are administered into the bloodstream. This specificcharacteristic permits them to be efficiently used in clinical trialswith a comfortable i.v. administration for the patient. (Douglas J T,and Curiel D T. Adenoviruses as vectors for Gene Therapy. Science andMedicine, March/April, 1997, 44-53).

[0025] Adenoviruses are double stranded DNA viruses. They have anicosahaedric structure, infect a great variety of mammalian cell types,and support the ubiquitous expression of a specific receptor in the cellsurface not yet identified. Its union to cells occurs by means of theprotein component of the capside and the virus enters into the cell byreceptor-mediated endocytosis.

[0026] More than 40 different human serotypes of adenovirus have beenidentified, of which type 2 (Ad2) and 5(Ad5) have been more extensivelystudied and, therefore, more widely used as vectors for gene therapy. Avery important characteristic of these two Ad serotypes is that theyhave never been associated with malignant human processes.

[0027] The strategy for the creation of recombinant adenovirus is basedon the organization of the adenoviral genome. The expression of theadenoviral genes occurs in two phases, early and late, that are definedby the time of replication of the adenoviral genome. The early genesencode themselves in 4 distinct transcriptional units: E1, E2 and E4encode for essential regulatory proteins that induce the replication ofthe adenoviral DNA. The gene E3 is a non-essential gene. The products ofthe late genes include the main proteins of the capside, which aretranscribed from a unique promoter. (Graham F L, and Van Der Eb A J. Anew technique for the assay of infectivity of human adenovirus 5 DNA.Virology 1973, 52:456-467).

[0028] The recombinant adenoviruses are generated by introduction of theexogenous gene or sequence of DNA of interest in substitution of theadenoviral genome regions required for the replication of the virus. Theadenoviral recombinant vectors present deletions in E1 and E3 genomeregions. Recombinant adenovirus generation Is conducted both through thereplacement of E1 or E3 regions or through the insertion of theexogenous gene between the E4 region and the right extreme of theadenoviral genome. Vectors based on the insertion of the exogenous genat the right extreme of the adenoviral genome or by the replacement ofthe E3 region maintain their replication capability. On the contrary,the substitution of early region E1 produces a faulty vector in itsreplication capability, that, therefore, can spread only in a cell linethat supplies in “trans” the absent functions of the replaced adenoviralregion, or in presence of a collaborator virus. Of these, the mostcommonly used as gene transference vectors are the replication-deficientadenovirus (Douglas J T, and Curiel D T. Adenoviruses as vectors forGene Therapy. Science and Medicine, March/April, 1997, 44-53).

[0029] The creation of adenoviral vectors, as well as their applicationfor the treatment of fibrosis, are shown in the examples describedhereinafter.

OBJECTS OF THE INVENTION

[0030] Hereinafter, the objects and advantages derived from thisinvention are presented.

[0031] An object of the present invention is to provide a procedure toprepare recombinant adenoviral vectors pAdGFP-MMP-8, by means of thecloning of the reporter genes: lac-7 and GFP and the therapeutic gene ofcollagenase or metalloprotease MMP-8 in its latent and/or active forms.

[0032] Another object of the invention is to provide an adenoviralrecombinant vector with an exogenous gene or DNA sequence of interestthat encodes for therapeutic proteins useful in the treatment of thegeneralized fibrosis, in target organs susceptible to suffer from it.Such genes are, but are not limited to MMP-8 active and latent, MMP-1,MMP-2, MMP-9 and MMP-13; and uPA (wild type and/or modified).

[0033] Also, in the present invention, pharmaceutical compositions areprovided which contain the recombinant adenoviral vectors in quantitiestherapeutically effective of viral particles for the treatment ofgeneralized fibrosis; as well as their uses and therapeutic applicationsin the treatment of fibrosis.

[0034] An advantage of greater importance in the treatment of thegeneralized fibrosis, particularly of hepatic cirrhosis, is that thedelivery of therapeutic genes is carried out through tissue-specificrecognition by the way of administration employed.

[0035] Another advantage of the therapeutic uses of the invention, whichis directed initially to revert hepatic cirrhosis, is the treatment ofgeneralized fibrosis in other target organs susceptible to suffer fromit, including, without limitation, the treatment of fibrosis in lung,heart, skin, kidney, among others, in mammalian animals, including humanbeings.

[0036] Another object is the design of a technology to send genesefficiently to livers of animals affected by cirrhosis that resemble twotypes of cirrhosis that usually affect human beings (Alcoholic cirrhosisand Primary Biliary Cirrhosis).

[0037] Another advantage resulting from the fibrosis treatment is thatrecombinant adenovirus does not induce lethal toxicity in none of theinjected animals with the vectors.

[0038] Another objective of the invention allows us to discriminate themodification of the staining reaction with X-Gal between the endogenoustissue -galactosidase activity and the bacterial -galactosidase inducedby the infectious action of the adenoviral vector. The use of the greenfluorescent protein permits us to verify the in vivo transduction ofdifferent organs in rats to verify if the vector administration wasappropriate, if the expression remains, and besides not killing theanimals it is possible to conduct follow up observation after surgery.

[0039] Finally, all this evidence let us suggest that our systemcomprises an efficient vehicle to deliver therapeutic genes such ashuman metalloproteases MMP-8 active and latent; MMP-1, MMP-2, MMP-9 andMMP-13; collagenase which degrade the deposited collagen excess and/orgenes which encode for promoters of hepatic regeneration such as humanuPA (urokinase Plasminogen Activator, modified and wild type),Hepatocite Grow Factor (HGF); the truncated receptor for TGF-β type IIand Smad 7 to livers of cirrhotic rats, with the purpose to re-establishnormal liver functions or normal functions of other organs affected bythe same pathology.

[0040] Thus, in the present invention a process of preparation is given,through which adenoviral recombinant vectors, pharmaceutical compoundsand therapeutic uses for the fibrosis treatment, especially for thetreatment of hepatic cirrhosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Other particularities and advantages of this invention will beevident in the following detailed description of the preferred objectsand embodiments, from the enclosed claims and from the drawings orshapes attached, in which:

[0042]FIG. 1 shows the cellular physiopathology of hepatic cirrhosis;

[0043]FIG. 2 shows the proof of concept on how gene therapy works byreverting the cirrhosis process;

[0044]FIG. 3 is the schematic representation, which shows the cloningand production of the adenoviral vector Ad5-gal;

[0045]FIG. 4 shows the schematic development of the AdEasy system togenerate recombinant adenoviruses, specifically the pAdGFP-MMP-8;

[0046]FIG. 5 shows the analysis of the expression of -galactosidase incultured cells.

[0047]FIG. 6 shows the expression determination of green fluorescentprotein (GFP) expression in cultured cells;

[0048]FIG. 7 shows the expression of -galactosidase in different organsafter the infusion with Ad5 gal through the iliac vein.

[0049]FIG. 8 shows the analysis of the tropism of the vector Ad5-gal todifferent organs of cirrhotic experiment animals by chronic intoxicationwith CCl₄, demonstrating that the main target organ is the liver;

[0050]FIG. 9 shows the analysis of the tropism of vector Ad5gal todifferent organs of cirrhotic experiment animals. Cirrhosis was inducedby bile duct ligation and it was demonstrated that the main target organis the liver.

[0051]FIG. 10 shows histological sections of representative images ofthe in vivo efficiency transduction assays of the vector Ad5-gal incirrhotic rats with chronic administration of CCl₄;

[0052]FIG. 11 shows histological sections of representative images ofthe in vivo efficiency transduction assays of the vector Ad5-gal incirrhotic rats by common bile duct ligation;

[0053]FIG. 12 shows the in vivo determination of the expression of thegreen fluorescent protein;

[0054]FIG. 13 shows the cloning strategy of the latent MMP-8 and activeMMP-8;

[0055]FIG. 14 shows the mechanisms of complex formation with DNA ofMMP-8s for in vitro transfection essays in cells of hepatic origin(HepG2);

[0056]FIG. 15 shows the verification through electrophoresis in agarosegels of the success of cloning of MMP-8 cDNAs in the appropriateplasmids;

[0057]FIG. 16 shows the transfection efficiency in HepG2 cells (Cells ofhepatic origin) with the plasmids of -galactosidase and cDNA-MMP-8;

[0058]FIG. 17 shows the analysis by polymerase chain reaction associatedto reverse transcriptase (RT-PCR) of MMP-8 messenger RNAs;

[0059]FIG. 18 shows analysis of the collagenolytic activity in theprotein secreted to the culture medium by HepG2 cells after transfectionwith cDNAs for latent MMP-8 and active MMP-8;

[0060]FIG. 19 shows the hormonal regulation of the MMP-8 gene expressionunder the transcriptional control of the regulable promoter PEPCK and,

[0061]FIG. 20 shows the dose-response assay of the different doses usedto determine the best response of “in vivo” hepatic transduction withthe -galactosidase reporter gene.

DETAILED DESCRIPTION OF THE INVENTION

[0062] There are many reports showing that through systemicadministration of recombinant adenoviral vectors (AdR) into healthyexperiment animals, a specific homing and highly preferential tropism ofthese vectors into the liver is observed. Up to now, it was not knownwhether the AdR were able to transduce cirrhotic rat livers. Aspreviously mentioned, hepatic cirrhosis is characterized by an increaseof fibrosis in the entire liver parenchyma, mainly around the centraland portal veins, creating a barrier which hampers the exchange ofmacromolecules between the sinusoid and the hepatocytes (Antoni P P,Hishack K G, Nayak N C, Poulsen H E, Scheuer P J, Sobin L H. Themorphology of cirrhosis: Definition, nomenclature and classification.Bulletin of the World Health Organization. 1977; 55:521-540; and ScottL. Friedman: The cellular basis of hepatic fibrosis: Mechanisms andtreatment strategies, The New England Journal of Medicine, 1993,Vol.328, No. 25:1828-1835), and this protocol was designed to verify ifeven in presence of this barrier, the exogenous genes could besystemically delivered to the cirrhotic liver.

[0063] Therefore, our hypothesis is that AdRs containing LacZ and GFP(green fluorescent protein) reporter genes are capable of transducinglivers of cirrhotic rats even if the lobular architecture of the liveris distorted.

[0064] Thus, we could sent to these livers therapeutic genes such ashuman metalloproteases or collagenases human MMP-8 active and latent,MMP-1, MMP-2, MMP-9 and MMP-3; human Urokinase Plasminogen Activator(uPA wild type and/or modified); the truncated receptor for TGF-β typeII and Smad 7, which degrade the excess of collagenic proteins depositedand/or prevent the exacerbated synthesis of collagenic proteins, as itis shown in FIGS. 2 and 18; and/or genes which encode for proteinsstimulating hepatic regeneration such as uPA, in order to re-establishthe normal functioning of the liver, as is shown in FIG. 2.

[0065] The current invention initiates a research line to carry out genetherapy as an alternative for the treatment of chronic degenerativedisease, specifically of hepatic cirrhosis in human beings, through theestablishment of an efficient vehicle to send genes to the liver whichwill produce therapeutic proteins to help re-establish the normalfunctions of the liver, see FIG. 2. FIG. 2 shows how sending efficientlya therapeutic gene to the liver, in this case, a collagenase(metalloproteases of matrix, MMPs), it is possible to promotedegradation of collagen through the over-expression of thesemetalloproteases.

[0066] In FIG. 3, the strategy for the cloning and production of anadenoviral vector is shown. The plasmid pDeltaE1sp1B contains adenovirusAd5 sequences, in which the bacterial gene Lac-z was inserted. Thisplasmid was recombined with the pBHG10 to obtain complete viralparticles after co-transfection in the cell line 293. The vector pAdGFPwas obtained as follows: the MMP-8 gene (coming from the plasmidPEPCK-MMP-8) was cloned in the vehicle vector, pAdTrack-CMV, theresultant plasmid is linearized with the restriction endonuclease Pme I,and is then transformed in E. coli (BJ5183) with the plasmid pAdEASY-1.The recombinant colonies were selected through kanamicine resistance,and the recombination is confirmed by restriction analysis withendonucleases. Finally, the recombinant plasmid linearized istransfected in the packaging cell line (293 cells), the recombinantadenoviruses are obtained within 7 to 12 days as illustrated in FIGS. 3and 4 (Tong Chuan H., Shibin Z., Luis T. Jian Y, Kenneth W. andVolgestein Bert: A simplified system for generating recombinantadenoviruses. Prod. Natl. Acad. Sci.USA Vol. 95: 2509-2514, March 1998).To evaluate the grade of transduction in vitro liver HepG2 cell line andperitoneal macrophages isolated from mouse were used. In FIG. 5 theexpression of -galactosidase in cultured cells is shown. A), B) and C)correspond to HepG2 cells (320×); D), E) and F), are mouse peritonealmacrophages (100×). In C) and F) the transduced cells are shown with1×10⁸ viral particles/ml from the Ad5-Gal vector. Three techniques wereconducted to compare the degree of incorporation of the reporter geneLac-Z which was administered to each culture dish in the form ofplasmidic DNA PGK-Gal, through precipitation with Ca⁺⁺ phosphate (ChenC, and Okayama H. Calcium Phosphate mediated gene transfer, a highlyefficient system to establish transforming cells with plasmidic DNA.Biotechniques 1988, 6:632-638), DNA complexes-polylysine-Lactose(Martinez-Fong D., Mullersman J E, Purchio A F, Armendariz-Borunda J.,and Martinez-Hernandez A., Non enzymatic glycosylation of poly-L-lysine:A new tool for targeted gene delivery. Hepatology, Vol. 20, No. 6:1602-1608), with the vectors -Ad5-gal and pAdGFP-MMP8. The visualizationof the activity of Gal was verified with the reactive Xgal and the GFPin a microscope-stereoscope of fluorescence. For the in vivo assay, galstaining was standardized using different pHs of the suspension with thereactive Xgal (Weiss D J, Ligitt D., and Clark J G. In situphotochemical detection of galactosidase activity in lung: assessment ofXgal reagent in distinguished Lac-Z gene expression and endogenousgalactosidase activity. Human being therapy, Sep. 1, 1997, 8:1545-1554).

[0067] The models of experimental hepatic cirrhosis used are: a) Chronicintoxication caused by carbon tetrachloride (CCl₄), in which hepaticcirrhosis is established starting from the 8^(th) week of peritonealadministration (Mion F, Geloen A, Agosto E. and Minaire Y. Carbontetrachloride induced cirrhosis in rats: influence of the acute effectsof the toxin on glucose metabolism. Hepatology 1996, Vol. 23, No.2:582-587); and B). ligation of the bile duct (LCB) in which cirrhosisis observed after the fourth week of surgery (Lee S, Giraud C., DraillonA., HADengue A., and Lebec D., Hemodynamic characterization of chronicbile duct ligated rats; effect of pentobarbital sodium. AM Journalfisiol. 1986; 251:176-180; Nakano S., Harakane J. and Hashimoto H.,Alteration in peribiliary ducts microcirculation in rats after commonbile duct ligation. Hepatology, 1995, Vol. 21, No. 5: 1380-1995; DumasWalla R., Belcowitz D., and H. Eubi J E. Adaptive response of theEnterohepatic circulation of bile acid to extra hepatic. CholestiasisHepatology 1996, Vol. 23, No. 3: 623-629 and Poo J. L., Stanes A.,Pedraza-Chaverri J., Cruz C., Pérez C., Huberman A. and Uribe M:Cronologia de la Hipertensión Portal, Disminución de la Excreción desodio y activación del sistema renina-angiotensina en cirrosis biliarexperimental. Rev., Invest Clin, 49:15-23,1997).

[0068] Ad5 gal was administered at the same time and from the same lotto control rats without cirrhosis. Rats with 5 and 8 weeks of CCl₄intoxication and rats with 2 and 4 weeks of bile duct ligation (BDL)were sacrificed 72 hrs after administration of recombinant adenovirusfor the histological analysis and determination of the expression of thegalactosidase protein (gal) encoded by the AdR. For this purpose liver,spleen, heart, lungs, kidneys and brain were extracted, tissue sectionswere cut in cube shapes of 5 to 6 mm., which were absorbed in freezemedium Tissue-Tek O.C.T., the tissues were frozen at −30° C. and theywere cut with a cryostat to obtain 8 μm sections These sections wereplaced on silanized glass slides and fixed with formaline, pH 8.5,during 15-30 minutes and were exposed to Xgal for 16-18 hours, beingcounterstained with Neutral Red stain. (Weiss D J. Ligitt D. and Clark JG. In situ Hiti Chemical Detection of galactosidase activity in lung:assessment of Xgal reagent in distinguishing 1AC-Z Gene expression andendogenous galactosidase activity. Human Gene Therapy, Sep. 1, 1997,8:1545-1554). The percentage of positive cells was determined bymorphometric analysis in multiple fields of the same size andcalculating the average. Besides, liver sections of cirrhotic rats wereobtained and tissues absorbed in paraffin were cut and stained withSirius red which specifically stains collagenic proteins(Armendariz-Borunda J., and Rojkind M., A simple quantitative method forcollagen typing in tissue samples: Its application to Human liver withschistosomiasis. Collagen Rel. Res 1984, Vol. 4, 35-47). Through thistechnique we can verify clearly the degree of fibrosis and the increaseof bile ducts in the hepatic parenchyma. To verify the in vivotransduction of cells with GFP, we used healthy Wistar rats thatreceived pAdGFP MMP-8 vector. 72 hours later, a laparotomy was performedand the exposed organs were visualised in the microscope offluorescence, closing the wound afterwards to keep the animal alive.

[0069] The previous results that are presented here regarding the studyof the physiopathology of experimental hepatic cirrhosis are summarizedin FIG. 2. Said figure shows the role of pro-inflammatory andpro-fibrogenic cytokines produced In vivo by Kupffer cells which, inturn, activate the hepatic stellate cells (HSC) to have them produceexcess collagens deposited in the subendothelial space, obstructing theexchange between hepatocytes and sinusoids (Armendariz-Borunda J.,Katayama K., and Seyer J. M.: Transcriptional mechanisms of type Icollagen gene expression are differentially regulated by IL-1beta,TNFalfa and TGF into cells. J. Biol. Chem. 267:14316-14321, 1992;Armendariz-Borunda J:, Katai H., Jones C. M. Seyer J. M. Kang A. H. andRaghow R.: Transforming growth factor beta is transiently enhanced at acritical stage during liver regeneration following CCL4 treatment.Laboratory Investigation. 69:283294, 1993 and Armendariz-Borunda J., RoyN., Simjewish C., Raghow R. Seyer J. M. and Kang A. H.; activation ofIto cells involves regulation of API collagen Gene Expression.Biochemical Jounal 304:817-824, 1994). The degree of incorporation ofLac-z gene in cultured cells showed visible differences betweentechniques of Calcium-Phosphate, DNA-polilysine-lactose complexes andwith the recombinant adenoviral vector in HepG2 and PMM (Peritonealmouse macrophages). The degree of transduction with adenovirus reaches100% and with the other two techniques about 1% as shown in FIG. 5. FIG.6 shows the expression of green fluorescent protein (GFP) in culturedcells. A). Peritoneal mouse Macrophage transduced with the adenoviralvector pAdGFP-MMP8, 72 hours after its administration (50×), B).HepG2cells transduced with the adenoviral vector pAdGFP-MMP8, 72 hours afterits administration (50×) and C). HepG2 cells without the adenoviralvector. All the pictures were taken In a microscope stereoscope offluorescence. It is necessary to point out that in the development toidentify galactosidase activity, the cells must be fixed and they die.In the GFP assay, the cells are still intact and alive.

[0070]FIG. 7 shows the expression of gal in different organs afterinfusion with Ad5 gal by iliac vein. Fixation, washing and Xgalsolutions using different pHs were used to discriminate among theendogenous expression and the bacterial exogenous galactosidase. Infigure A, a pH 7.0 was used and in Figure B the pH was 8.5. This is thesummary of the results of the assays of the different experimentalconditions and it can be appreciated that the tissue exposition to Xgalsolution with a pH 8.5 allowed us to eliminate the expression ofendogenous galactosidase. We obtained frozen tissue sections fromdifferent organs: liver, kidney, lung, heart, brain and spleen fromnormal rats and intoxicated with CCl₄ for five and eight weeks. Asrepresented in FIG. 8, the graphics show clearly that the main targetorgan is the liver, both in healthy rats as well as in rats with chronicadministration of CCl₄. A) 5 weeks of CCl₄ administration and B) 8 weeksof CCl₄ administration. Spleen and lung present a degree of trasductionbelow 1%, and thus this is not evident from the graphs. Rats receiveddoses of 3×10¹¹ viral particles/ml of Ad5gal vector. The healthy controlrats presented a total of 70% of hepatocytes transduced, while spleenand lung showed less than 1% transduction. In the other organs notransduction was found. Tissue sections were obtained from healthy ratsas described before and compared with tissues from rats with 2 and 4weeks of BDL. FIG. 9 clearly shows how the main target organ is theliver, both in healthy rats as well as in BDL rats. A) 2 weeks of LCBand B) 4 weeks of BDL. The spleen and the lung present a transductiongrade lower than 1%, and thus it is hardly noticeable in graphs. With adose of 3×10¹¹ viral partides/ml of the AD5gal vector, BDL rats presenta total of 10% transduced hepatocytes. Besides liver, spleen and lungpresented less than 1% transduction. The other organs showed notransduction. In FIG. 10, histological results are shown with thehepatic cirrhosis model induced by the chronic administration of CCl₄,where A) represents a liver section of a normal rat, 72 hours after theadministration of Ad5 gal, by iliac vein (one representative cut of theexperiments of a total of 5 rats). More than 70% of the hepatocytes arepositive to the expression of gal (200×); D) The same liver as in FigureA, but stained with Sirius Red to observe collagen synthesis anddeposition (200×); B) liver with 5 weeks of chronic intoxication withCCl₄. About 30-40% of the hepatocytes were successfully transduced; E).The same livers as in B, but stained with Sirius Red, the increase inthe amount of collagen is notable and the liver architecture begins todistort (200×); C) rat liver after 8 weeks of chronic intoxication withCCl₄ to cause cirrhosis, again more than 40% of liver cells werepositive for βgal expression and F) the same livers as in C, but stainedwith Sirius Red. Large deposits of collagen formed between the centraland portal veins (200×) are characteristic. In FIG. 11, results obtainedin the model of biliar duct ligation (BDL) induced cirrhosis are shown.A) shows a liver section of a normal rat 72 hours after theadministration of Ad5 gal, by iliac vein (one representative cut of theexperiments of a total of 5 rats). More than 70% of the hepatocytes arepositive to the expression of gal (200×); D) the same liver as in FigureA, but stained with Sirius Red to observe collagen (200×); B) rat liverafter 2 weeks of BDL. β-gal essay was conducted 72 hours after Ad5βGaladministration, via iliac vein. About 10% of the hepatocytes weresuccessfully transduced with the reporter gen; E) the same livers as inB, but stained with Sirius Red. Liver architecture begins to distort dueto colestasis-induced fibrosis as well as to the important increase ofbiliar ducts (200×); C) rat liver after 4 weeks of BDL to causecirrhosis. β-gal essay was conducted 72 hours after the administrationof Ad5βGal, via iliac vein. Again, 10% of hepatocytes were successfullytransduced and F) the same livers as in C, but stained with Sirius Red.Observe the large deposit of collagen proteins formed as well as theproliferation of biliar ducts (200×). FIG. 12 shows a laparotomy of ahealthy Wistar rat that received pAdGFP-MMP-8 vector. The expression ofthe GFP is clearly seen in the liver and in insignificant amounts in thespleen. A very important fact is that the injection of adenoviralvectors did not induce lethal toxicity in experiment animals, bothhealthy and controls.

[0071] The preferred way to apply the present invention is throughendovenous administration of the recombinant adenoviral vectors of thisinvention or the pharmaceutical compound which contains them, in whichtherapeutically effective amount is administered with an unitary doseregimen convenient to an individual with fibrosis. This regimen can beadjusted according to the affliction degree. Generally, unitary doses ofabout 10⁷ to 10¹⁴ viral particles for individual are employed. Thepreparation of a pharmaceutical compound including the adenoviralrecombinant vectors of this invention can be conducted through theemployment of standard techniques very well known by the persons skilledin the art, in combination with any of the pharmaceutically acceptablecarriers described in the state of the art, including withoutlimitation, starch, glucose, lactose, sacharose, gel, malt, rice, wheatflour, chalk, silica-gel, magnesium stearate, sodium stearate, powder ofglyceril monostearate, NaCl, glycerol, propilene glycol, water, ethanol,and similar. These compounds can take the pharmaceutical form ofsolutions, suspensions, pills, tablets, capsules, powders and slowrelease formula, and similar.

[0072] The above description and the following examples have the purposeto illustrate particular embodiments of the invention and they shouldnot be considered as limitations of the scope of this patent.

EXAMPLES Example 1 Methodology to Demonstrate the Activity ofMetalloprotease or Collagenase (MMP-8) and How to Regulate its Function

[0073] a) Cell culture. HepG2 cells is a cell line of parenchymal originderived from a human hepatoma, and were cultured in 60 mm culture dish,37° C. in a wet atmosphere, with 95% air and CO₂ 5% atmosfere in Eagle'smedium modified by Dulbecco (DMEM), supplemented with 10% fetal bovineserum, 2 mM L-Glutamax and antibiotics (100 U/ml penidillin and 100g./ml. streptomycin).

[0074] b) Vectors of Expression of Latent and Active MMP-8 Genes

[0075] Two plasmids were used with 2 kinds of MMP-8 genes to transfectthe hepatic cells: The plasmid pcDNA-MMP-8 which contains the cDNA whichencodes for latent MMP8 (pro-MMP8) together with the strong viralpromoter of cytomegalovirus (CMV); and the plasmid pcDNA3MMP-8containing the cDNA which encodes for the active MMP-8, together withthe CMV promoter. This last one was created through subclonation usingpcDNA₃ and PETIIa-HNC plasmids, cutting with the restriction enzymesBamHI and XbaI and inserting the PCR product coding for the MMP-8catalytic domain (which lacks the propeptide and carboxi-terminalfragments), as shown in FIG. 13, the delivery of latent and active MMP-8genes. Two types of plasmids with the MMP-8 gene were used to bedelivered to hepatic cells in culture: 1) PcDNA₃-MMP-8, plasmid with thestrong viral promoter of the cytomegalovirus (CMV) and the cDNA which encodes for the collagenase in its active form.

[0076] As a reporter gene pSV₂-gal plasmid was used. Said plasmid hasthe gene which encodes the enzyme -galactosidase inserted adjacent tothe SV40 virus promoter.

[0077] c) Plasmid Transformation, Amplification and Purification

[0078] To obtain a large enough quantity of each one of the plasmids tobe used in the various assays, each plasmid was introduced to E. coliDH5™, (this process is known as transformation), according to theinstructions of the supplier. (Life Technologies, Gaithersburg, Md.): ina reaction tube 50 l of the competent strain DH5 were used and 2 l ofplasmids (1-10 ng of DNA) were added. After mixing, it was incubated onice during 30 minutes, a thermal shock (37° C. for 20 seconds) wasapplied and it was immediately chilled on ice for 2 minutes. At the endof this period of time, 0.95 ml of the bacterial culture medium LuriaBase (LB) was added and it was stirred at 225 rpm during one hour to 37°C. to allow plasmid expression. After the expression, 50 l of thereaction mix were taken and seeded onto an Agar plate with 100 g/ml ofampiciline and it was incubated to 37° C. overnight. The colonies thatgrow after this period are those which contain the plasmid of interest,because of the resistance against the antibiotic.

[0079] To amplify the plasmid, two colonies were taken from the Agarplate and grown in a liter of LB medium containing 100 g./ml ofampiciline during 24 hours at 37° C., with constant stirring at 225 rpm.Once the optic density of the culture was 0.6, it is centrifuged to6,000 rpm for 20 minutes to recollect the bacterial pellet. From thisbacterial pellet, plasmidic DNA was separated from the genomic DNA ofthe bacteria using a kit of plasmids purification (Monster-prep, BIO101,Vista, Calif.), which is based on the alkaline lysis of the bacterialwall, the liberation of the plasmid in its interior and the separationof this DNA through a particular resin. The quantification of theplasmidic DNA was performed measuring spectrophotometrically theresultant absorbance at λ=260 nm.

[0080] d) Transfection of cultured cells

[0081] One of the most commonly used methods to introduce genes toeucaryotic cells, is DNA transfection with calcium phosphate, in whichthe exogenous DNA is precipitated as a fine complex on the cell surface,to be later incorporated by the cell and transiently integrated in thechromosomal DNA. To deliver the DNA with more selectivity to the hepaticcells, DNA is used in the form of complex with polylysine-lactose,because of hepatic cells have a specific receptor for Galactose in theircell membrane. For this, HepG2 cells were cultured at 70-80% confluenceand then transfected with plasmids pcDNA-MMP-8, pcDNA₃-MMP-8 andpSV₂-galactosidase. Transfection was carried out by DNA precipitationwith calcium phosphate (Graham, and Van derEb, 1973; Chen and Okayama1988) and by complex formation with polylysine-lactose (Martinez-Fong etal, 1994). Briefly, cultured cells were added with the newly formedprecipitate, product of the addition to plasmidic DNA of a solution ofDNA with CaCl₂ 2M, in buffer solution HEPES. pH 7.12 in case of thetransfection with calcium phosphate or DNA complex withpolylysine-lactose is added. Cells are incubated from 4-16 hours toallow the precipitate to appear to the cell surface, and later the DNAcan be endocyted and introduced transiently to the nucleus. At the endof this time, the culture medium is replaced for a fresh one, see FIG.14, where HepG2 cells are cultured with DMEM medium with 10% bovinefetal serum. When 60-80% confluency is reached, 10 mg of plasmid withMMP-8 gen is added in its latent form, as well as in the active ormature form. At the same time, the prokaryotic gene of galactosidase(-gal). is added to monitor the transfection and expression efficiency.MMP-8 gene was sent in different forms: naked, in complex with CaPO₄ orin complex with polylysine-lactose.

[0082] e) Formation of Complexes Polylysine-Lactose and DNA:Polylysine-Lactose (DNA:PL)

[0083] The polylysine-lactose complex is formed when 14.8 mg ofpoly-L-Lysine (0.1 N) react with 200 l of -lactose 0.5 N(lactose-polylysine ratio: 1.0 N). Then, 20 mg of reducing agent sodiumcyanoborohydride 3 M is added and it Is incubated at 37° C. for 48 hourswith constant stirring at 225 rpm. Then, the reaction goes through adesalting column (BioRad 10-DG) previously conditioned with phosphatebuffer (PBS pH 7.2), which is eluted with the same buffer. Carbohydratecontent is determined to the eluted fractions by the method of DuBois(1956) to analyze the degree of lactosylation of the complex and thecontents of polylysine according the method of Shen et. al. (1984),which is considered as a base to evaluate the final concentration of thePL complex. The fraction with a mayo. concentration of PL is used forits further reaction with the DNA of the plasmid containing the gene ofinterest, as is shown in FIGS. 14 and 16.

[0084] To evaluate the optimal molar ratio of DNA: PL to be used intransfection assays, the DNA was made to react with severalconcentrations of PL. At the end of one hour of incubation, samples wereapplied to a 1% retardation agarose gel and submitted to electrophoresisof DNA (60 millivoltios, 1.5h), in which the DNA: PL complex with thelargest PL contents runs a shorter distance than the one run by the freeplasmid (0% retardation). The DNA: PL ratio which causes 80 to 90% ofretardation of migration in the agarose gel was used as shown in FIG. 16to obtain an efficient expression of exogenous genes of -galactosidaseand pcDNA-MMP-8 delivered to HepG2 cells in complexes with CaPO₄ andpolylysine-lactose.

[0085] f). Assays of Transient Expression Using the Reporter gen Systemof -Galactosidase(gal)

[0086] This system determines the activity of the -galactosidase enzymeas a measure of the level of expression of the transfected gene ofinterest along with Lac Z gene which encodes for this enzyme. Thegalactosidase is a bacterial enzyme which catalyzes the conversion ofthe uncolored substrate X-gal to a product of blue coloration. Becauseof this, the -galactosidase activity observed in eucaryotic cellssubjected to transfection will indicate the successful incorporation ofthe gene of interest associated to the bacterial gene.

[0087] The assay of -gal for the stain of cells in culture dish consistsin the fixation of cells at 4° C. during 5 minutes with 2%p-formaldehyde, the subsequent wash with PBS (3×) and the addition ofone ml of a stain solution in PBS containing 20 mM potassiumferricianide, 20 mM potassium ferrocianide and 2 mM Magnesium Chloridefollowed by the addition of the substrate Xgal in a final concentrationof 0.5 mg/ml. After incubation at 4° C. overnight (18 hours) blue cellsare identified under the microscope (Ausubel, 1995).

[0088] g) RNA Extraction

[0089] 48 hours after transfection, cells are recollected to extract RNAby the Method of Chomczynski and Sacchi (1987) using the reactive ofTrizol™, as described hereinafter: to each one of the cell dishes one mlof PBS solution was added and cells were recollected by scraping themfrom the dish and transferred to an Eppendorf tube. It was thencentrifuged at 1000 rpm for one minute and the cell pellet was treatedwith 500 l of Trizol, homogenized and incubated for 5 minutes at 4° C.One hundred μl of chloroform were added, and incubation was conductedduring 5 minutes at 4° C. After this, it was centrifuged at 12,000 g for15 minutes at 4° C. and the aqueous upper phase was transferred to aclean tube in which an equal volume of isopropanol is added andincubated at −70° C. during 15 minutes to precipitate the extracted RNA.Then, it is centrifuged at 12,000 g during 15 minutes at 4° C., thesupernatant is eliminated through decantation and grying the tube withclean and sterile paper. Then, 500 l of 75% ethanol were added and itwas centrifuged at 12,000 g during 10 minutes to 40° C. Finally, the RNApellet was resuspended with 20 to 50 l of deionized water treated withdiethylpirocarbonate (DEPC) and RNA concentration was quantified byspectrophotometry at λ=260 nm.

[0090] h) Analysis of Expression of MMP-8 Gene by the Polymerase ChainReaction (PCR) Associated to the Reaction of Reverse Transcriptase(RT-PCR).

[0091] To determine the degree of expression of the exogenous gene ofMMP-8 incorporated to the cell, complementary DNA was obtained (cDNA)starting from RNA previously extracted and then amplifying theexpression signal by the Polymerase Chain Reaction.

[0092] To obtain the cDNA, the following procedure was used: 2 g oftotal RNA were taken to a volume of 8 l with deionized, sterilized waterand incubated at −70° C. for 10 minutes. Then, the sample was stirred iniced water during 5 minutes and still in the ice, the following reagentswere added: 4 l of 5× buffer for the RT enzyme, 4 l dNTP's mix 2.5 mM, 1l random primers (1 g/l), 1 l inhibitor of RNAase (one U/l) and finally2 l of the Reverse Transcriptase enzyme (200 U/l). The reaction mix wasincubated at room temperature for 10 minutes and then at 37° C. for onehour. At the end of this time, it was placed immediately in atemperature of 95° C. for 10 minutes, and then it was placed on icedwater during 5 minutes with constant stirring and it was stored at −70°C. until its further use.

[0093] To analyze the specific expression of MMP-8 gen, a PCR reactionwas set up using the primers or oligonucleotides specific for this geneaccording to the experimental conditions described hereinafter: in areaction tube containing 2 l of cDNA 5 l of 2.5 mM MgCl₂, 5 l 5× bufferfor the polimerase enzyme, from leukemia murine virus of Moloney (MMLV),l of 2.5 mM dNTPs, 5 l of the sense primer 3 μM, 5 l of the antisenseprimer 3 μM, 1 l of the polymerase enzyme (U/l) and it is taken to afinal volume of 50 I with deionized water (Innis et al, 1990). Theoligonudeotide sense primer specific for MMP-8 is5′-AGCTGTCAGAGGCTGGAGGTAGAAA-3′, and the antisense primer is5′-CCTGAAAGCATAGTTGGGATACAT-3′ (Cole et al., 1996). After the additionof these reagents, the mix was placed in a thermalcycler during 30cycles according to the following program: denaturation (94° C., 5 min),annealing (60° C., 1 min.) and extension (72° C., 1.5 min). Then, PCRproducts are submitted to electrophoresis (60 mV, 1.5 h) in a 1.5%agarose gel.

[0094] i) Assay of Collagenase Activity.

[0095] The analysis of enzymatic activity of collagenase was performedto determine the functionality of the enzyme produced, because thisprotein could be found enzymatically inactive, even when RNA expressionwas positive. Cells are cultured in serum-free medium for 24 hours,culture medium is recollected and activity of collagenase secreted bythe cells is determined by a modified method of Hasty et al. (1986) toidentify products of degradation of specific collagen substrate through8% polyacrylamide gel electrophoresis.

[0096] Briefly: cell supernatants containing 1-1.5 gr of protein wereincubated at 27° C. during 18 hours with 5 g of native collagen type Iand 60 l of the incubation buffer: 50 mM Tris-HCl, 5 mM CaCl₂ 0.02%NaN₃, 50 mM arginine, 1% Triton X-100 and in absence or presence of 1 mMAPMA, pH 7.6. Finally, 30 l of product of reaction were mixed with 30 lof sample buffer for proteins and electrophoresis in SDS-polyacrilamidegels (7.5%) was run to identify the degradation products 1^(A) and 2^(A)of collagen type 1.

Example 2 Results to Demostrate the Activity of Metalloprotease orCollagenase (MMP-8) and Therefore to Regulate its Function

[0097] Subcloning permitted to incorporate MMP-8 cDNA encoding for thefully functional enzyme was subcloned in a vector appropriate to ourneeds. Thus, FIG. 15 shows an electrophoresis of the DNA fragmentsreleased by cutting MMP-8 plasmids with restriction enzymes. Lane A).Marker of bp of 1 Kb DNA ladder (Gibco BRL); B). Perfect DNA marker(Novagen, Inc.); 1) pcDNA-MMP-8 cutting with BamHI and XbaI; 2)pcDNA3-MMP-8 cutting with BamHI and XbaI; C) φX174 marker (Gibco BRL); λHind III Marker (Gibco BRL), in which the latent MMP-8 cDNA (lane 1) andthe mature MMP (lane 2); were successfully subcloned in the expressionvectors pcDNA and pcDNA3. The released inserts are observed aftertreatment with restriction enzymes BamHI and XbaI. The bands stainedwith ethydium bromide correspond to each of cDNA (between 506 and 560base pairs) for mature and latent MMP-8 cDNA, respectively. To evaluatethe efficiency of incorporation of the cDNA for MMP-8 delivered to HepG2cells in form of complex with CaPO₄ and with polylysinelactose, theco-transfection of this plasmid was realized along with the reportergene of -galactosidase. In this way, cells observed in the microscopewith blue staining, indicate indirectly that they have also incorporatedto the plasmid of interest. FIG. 16 shows the expression of-galactosidase in HepG2 cells, co-transfected with free plasmid, in formof complex with CaPO₄, or in its form of complex withpolylysine-lactose. This figure shows that the DNA binding withpolylysine-lactose was accomplished because the higher the polylysineconcentration, the dearer the retardation of -gal plasmid. The ratioselected to transfect the cells was the one that delayed 80% of plasmidmigration.

[0098] Once demonstrated that the cells in culture are capable ofincorporate and express genes that have been transfected, it wasnecessary to corroborate that such genes were transcribed by themachinery of host cells by means of RT-PCR assays. FIG. 17 shows ananalysis by RT-PCR of messenger RNA for MMP-8 and MMP-13. (This plasmidwas used as a further positive control of transfection); in which a DNAelectrophoresis of PCR amplified products, of the cDNA for MMP-8delivered as a complex with CaPO₄ and polylysine-lactose, has beentranscribed for both cases in transfected HepG2 cells. It is observedthat product signal of PCR of MMP-8 (359 base pairs), was more intensewhen plasmid was delivered as a complex with polylysine-lactose.

[0099] To demonstrate that MMP-8 transcripts expressed by HepG2 cellswas translated into a functional protein, the assay for enzymaticactivity was conducted, using collagen type I as substrate. FIG. 18shows the enzymatic activity of type I collagen degradation of theprotein secreted in the culture medium, which was observed in thetransfected cells with the gene of latent MMP-8. With previousactivation with the mercurial agent APMA (lane 7) and with the gene ofactive MMP-8 complexed with CaPO₄ (lane 9) and with polylysine-lactose(lane 10), and its specific inhibition with EDTA 2 mM. Negativecontrols: type I collagen without addition of supernatants of cells(lane 1) and with addition of Trypsin (lane 3), collagen withsupernatants of cells without transfection (lane 2). Positive controls:type I collagen with supernatant of human leukocytes (lane 3), type Icollagen with addition of 0.015% bacterial collagenase (lane 4); anddegradation products of native type I collagen, separated in a 6%polyacrylamide gel, after it was incubated with supernatant oftransfected cells with latent and active MMP-8 genes. It was observedhow in both cases the collagenolytic activity is clear in presence ofAPMA in the case of latent MMP-8, and its inhibition for EDTA for bothlatent and active MMP-8. This fact shows that this proteolytic activitycorresponds to a metalloprotease of interstitial matrix. The incubationof native type I collagen with trypsin did not show degradation. So,this experiment clearly shows that MMP-8 action was specific consideringthe intact nature of the collagen molecule.

[0100]FIG. 19 shows evidence that activities of the enzymes thatspecifically degrade collagen can be controlled (turned off and/orturned on) through the cloning of its respective cDNAs that arethemselves under the transcriptional control of promoters of regulablegenes, such as the PEPCK (Phosphoenol-piruvate carboxikinase) gene. Itis clear that both the stimulation of cells in culture with Glucagon(lanes 5 and 6), and cyclic AMP (lanes 7 and 8), up-regulate theirproduction of messenger RNA that codes for MMP-8. It Is also clear thatInsulin lowers said production (lanes 9 and 10).

[0101] The observations regarding the activity of endogenous-galactosidase suggest that this activity is usually granular and weakerin color than the dark blue as a result of the activity of exogenousenzyme (Shimohama S., Rosenbergh M B. Fagan A M, Wolff J A, Short M P,Bradfielf X O, Friedman T., and Gage F H: Genetically Modified Cellsinto the rat brain: Characteristics of E. coli-Galactosidase as areporter gene. Brain Res. 5:271-278, 1989). Many modifications have beendescribed to increase the specificity in the determination of exogenousLac Z gene essay. Thus, according to previous information by Weiss, D J,Ligitt D., and Clark J G. In situ histochemical detection ofbeta-galactosidase activity in lung. Assessment of Xgal reagent indistinguishing Lac Z gene Expression and endogenous -galactosidaseactivity. Human Gene Therapy, Sep. 1, 1997, 8:1545-1554; in the presentinvention a solution of X-gal, with a pH 8.5 was used; in this way, theactivity of exogenous -gal was demonstrated, minimizing the endogenousactivity in vivo.

[0102] One of the indicators actually used for in vivo monitoring theefficiency and location of transduced cells with recombinantadenoviruses, is the detection of green fluorescent protein (GFP)expression. For this purpose, the gene which encodes for this protein issubcloned in adenoviral vectors, and then through the use of afluorescent microscope, the fluorescence given by GFP can be observeddirectly without sacrificing the experiment animal which received thevector (Rojas-Martinez, A, Wyde P R, Montgomery Calif., Chen S H, Woo SL C and Aguilar-Cordova E.: Distribution toxicity and lack ofreplication of an E1A-recombinant adenoviral vector after systemicdelivery in the cotton rat. Cancer Gene Ther. 1998, y TongChuan H.,Shibin Z., Luis T., Jian Y., Kenneth W., and Vogelstein Berth: Asimplified system for generating recombinant adenoviruses. Proc. Natl.Acad. Sci. USA Vol. 95:2509-2514, March 1998). A large body of data hasbeen obtained that shows that, after the i.v. administration ofadenoviruses in healthy animals, the main target cells were hepatocytes.This has been observed in mice, rabbits, dogs and primates (Zem A M. andKresina T F, Hepatic drug delivery and gene Therapy. Hepatology 1997,vol. 25, No. 2, 484-491), but not in cirrhotic rats. Probably, theinjection in portal vein could be more efficient to get to the targetcells in the liver, providing them a favorable innoculum of viralparticles to the entire liver before being diluted into the bloodstream.This route is efficient, but it has the disadvantage that it requires alaparotomy. On the other hand, peritoneal administration is a faster andsimpler infusion, but it does not promote hepatocyte transduction. Theresults of the present invention show that the injection of 3×10¹¹ viralparticles by iliac vein in normal Wistar rats of approximately 200 g.produces a very high level of expression (70% of transducedhepatocytes). Our results are consistent with a previous report in whichspecific delivery of reporter genes in primates by saphenous veinproduced almost the same level of transduction and expression of thetransgene in the liver, as compared with infusion through portal vein(Marie Jean T F D, Poeters V., Lieber A., Perkins J., and Kay M A.Methods for multiple portal vein infusion in mice: Quantitation ofadenovirus-mediated hepatic gene transfer. Biotechniques February 1996,20; 278-285 and Zhu G. Nicholson A G. Zheng X., Strom T B, and Sukhame VP. Adenovirus mediated -galactosidase gene delivery to the liver leadsto protein deposition in kidney glomeruli. Kidney international. 1997,Vol. 52, 992-999). Furthermore, the expression of the reporter gene inour animals with cirrhosis induced by chronic administration of CCl₄ wassurprisingly almost as high as the normal rats (40% of transducedhepatocytes). These results are very exciting because our cirrhoticanimals could hardly survive the surgical procedure required toadministrate the adenovirus by the portal vein. This is due to alteredfunctional hepatic tests, and elevated prothrombin time as well asimportant bleeding. Although rats with bile duct ligation showed asubstantial reduction in the number of transduced hepatocytes (5-10%),it is also important the number of hepatocytes, which eventually couldbe transduced with therapeutic genes, such as metalloproteases (MMP-8)and/or genes which encode for stimulating proteins for hepaticregeneration such as uPA (Urokinase Plasminogen Activator) and Smad 7.

[0103] Other embodiments will be evident for people skilled in the artbased on the present description. Said embodiments are included withinthe true scope and spirit of the invention.

[0104] The definitions of the symbols used in the figures correspondingto the present invention, are shown below:

[0105]FIG. 1:

[0106] CEH=stellate hepatic cell.

[0107] CES=Endothelial sinusoidal cell.

[0108] CK=Kupffer Cell.

[0109] ESET=Subendothelial space.

[0110] HE=Hepatocytes.

[0111] HIDC=Liver with chronic damage.

[0112] HN=Normal liver

[0113] SINU=Sinusoid

[0114]FIG. 2:

[0115] COLASA=Coliagenase

[0116] DCA=Degradation of collagen.

[0117] TGE=Experimental gene therapy

[0118] MMPs=Metalloproteases

[0119]FIG. 3:

[0120] CT293=Co-transfection in cells 293

[0121] PG CsCl=Purification with CsCl gradients

[0122]FIG. 4:

[0123] BD=Right arm.

[0124] Bl=Left arm.

[0125] CTBK=Co-transfection in bacteria and selection in Kanamicine.

[0126] CUL=Culture

[0127] Ll PacI=Linearize with Pac I.

[0128] Ll Pmel=Linearize with Pme I

[0129] PV=Viral particles

[0130] T293=293 Cell transfection

[0131] GENADR=Generation of recombinant adenovirus.

[0132]FIG. 7:

[0133] B=Spleen.

[0134] CE=Brain

[0135] CO=Heart

[0136] % CT=Percent of transduced cells

[0137] H=Liver

[0138] P=Lung

[0139] R=Kidney

[0140] Sad -gal=Without the Ad -gal vector

[0141] CAD -gal=With the Ad-gal vector

[0142] X-GAL7=Reactive Xgal, pH 7.0

[0143] X-GAL 8.5=Reactive X-gal, pH 8.5

[0144]FIG. 8:

[0145] B=Spleen.

[0146] CCl45=5 weeks of intoxication with CCL4

[0147] CCl48=8 weeks of intoxication with CCL4

[0148] CE=Brain

[0149] CO=Heart

[0150] % CT=% of transduced cells

[0151] H=liver

[0152] P=Lung

[0153] R=Kidney

[0154] PV=Viral particles

[0155] HN=Normal Liver

[0156]FIG. 9:

[0157] B=Spleen

[0158] CE=Brain

[0159] CO=Heart

[0160] % CT=% of transduced cells

[0161] H=Liver

[0162] LCB2S=2 weeks of bile duct ligature

[0163] LCB4S=4 weeks of bile duct ligature

[0164] P=Lung

[0165] R=Kidney

[0166] PV=Viral particles

[0167] HN=Normal Liver

[0168]FIG. 13:

[0169] PROT=Protein

[0170] APMA: MERCURIAL AMONOPHENYL ACETATE

[0171]FIG. 14:

[0172] ACT -gal=-galactocidase activity

[0173] CES=Cells

[0174] EAC=Enzymatic Activity

[0175] PL=Polylysine

[0176] PROT=Protein

[0177] RGAL=Galactose residues

[0178] SNAD=Supernatant

[0179]FIG. 16:

[0180] ADND=Naked DNA.

[0181] GELRADN-PL=Retardation gel for polylysine

[0182]FIG. 18:

[0183] CA=With APMA.

[0184] CACE=With APMA and EDTA.

[0185] CaPO4=Phosphate.

[0186] CE=With EDTA.

[0187] COB=Bacterial Collagenase.

[0188] COL1=Type I Collagen

[0189] PL=Polylysine

[0190] SA=Without APMA

[0191] SNL=Leucocyte Supernatant

[0192] ST=No-transfected

[0193] TRIP=Trypsin

[0194]FIG. 20:

[0195] % CT=% of transduced cells.

[0196] PV=Viral particles.

1 2 1 25 DNA artificial sense primer for MMP-8 1 agctgtcaga ggctggaggtagaaa 25 2 24 DNA artificial anti-sense primer for MMP-8 2 cctgaaagcatagttgggat acat 24

1. A recombinant adenoviral vector which contains an adenoviral genomefrom which the open reading frames E1 and/or E3 have been deleted, butretains enough sequence to make the adenoviral vector able to replicatein vitro, said vector also contains a therapeutic gene or a DNA sequenceof interest regulated by ubiquitous promoters and/or tissue-specificpromoters that encodes for therapeutic proteins useful in fibrosistreatment of the fibrosis.
 2. The recombinant adenoviral vectoraccording to claim 1, in which the specific tissue-promoter is PEPCK. 3.The recombinant adenoviral vector according to claim 1, in which thetherapeutic gene or the DNA sequence cloned in such adenoviral vector isselected from latent and active human metalloprotease gene MMP-8, MMP-1,MMP-2, MMP-9 and MMP-13; human urokinase Plasminogen Activator gene (uPAwild type and/or modified), gene of the truncated receptor for TGF-βtype II; and Smad 7 which encode for therapeutic proteins, that degradeexcess of collagenic proteins deposited in the cirrhotic organs.
 4. Therecombinant adenoviral vector according to claim 3, in which thetherapeutic gene is a DNA sequence selected from the gene of theHepatocyte Growth Factor (HGF), which encodes for proteins stimulatorsof hepatic regeneration with the purpose to re-establish the normalfunctions of the liver.
 5. The recombinant adenoviral vector accordingto claim 1, in which the therapeutic proteins for the treatment offibrosis are the latent and/or active protein MMP-8, MMP-1, MMP-2, MMP-9and MMP-13; uPA wild type and/or modified; the truncated receptor forTGF-β type II; betaglycan; HGF and Smad
 7. 6. The recombinant adenoviralvector according to claim 1, which contains also the delivery oftherapeutic genes or DNA sequences which encode for therapeutic proteinsintended for the treatment of fibrosis in cirrhotic liver.
 7. Therecombinant adenoviral vector according to claim 6, in which thedelivery of the therapeutic genes Is carried out in other organs withgeneralized fibrosis.
 8. The recombinant adenoviral vector according toclaim 7, in which the tissue-specific recognition of the therapeuticgenes to the organs with fibrosis, is conducted by the administrationroute used.
 9. The recombinant adenoviral vector according to claim 8,in which the administration route is endovenous.
 10. The recombinantadenoviral vector according to claim 6, in which the organs withfibrosis are selected from liver, lung, heart, kidney, skin, andhypertrophic scars.
 11. The recombinant adenoviral vector according toclaim 10, in which the main target organ is the liver.
 12. Recombinantadenoviral vectors according to claims 1 to 11 in which the delivery oftherapeutic genes is realised through the use of viral or non viralvectors.
 13. The recombinant adenoviral vector according to claim 12, inwhich non viral vectors are selected from plasmids and cationic andanionic liposomes.
 14. The recombinant adenoviral vector according toclaim 6, in which the efficient sending of collagenase gene MMP-8 tocirrhotic liver, can induce the degradation of collagen by means ofover-expression of metalloproteases.
 15. The recombinant adenoviralvector according to claim 1, characterized because it is used for thetreatment of the hepatic, pulmonary, renal, heart fibrosis, keloids andhypertrophic scars, and which does not induce lethal toxicity.
 16. Aprocess to prepare recombinant adenoviral vectors through the cloning ofreporter genes Lac-Z and GFP and the therapeutic gene, which encodes fortherapeutic proteins for the treatment of hepatic, pulmonary, renal,and/or heart fibrosis, keloids and hypertrophic scars.
 17. The processaccording to claim 16 in which the therapeutic gene is selected from thehuman metalloprotease gene MMP-8 latent and active, MMP-1, MMP-2, MMP-9and MMP-13; gene for human uPA wild type and/or modified; Smad 7 andgene of the truncated receptor of TGF-β type II.
 18. The processaccording with claim 16 in which the recombinant adenoviral vector ispAdGFP-MMP-8.
 19. A pharmaceutical composition containing atherapeutically effective amount with a regimen of unitary doses ofviral particles of recombinant adenoviral vectors, according to claim 1,for the treatment of hepatic, pulmonary, renal, and heart fibrosis,keloids and hypertrophic scars, combined with a pharmaceuticallycompatible carrier.
 20. The pharmaceutical composition according toclaim 19, in which the unitary dose is of about 10⁷-10¹⁴ viral particlesfor an individual with fibrosis.
 21. The use of recombinant adenoviralvector according with claim 1, for the elaboration of a bio-medicationfor the treatment of hepatic, pulmonary, renal, and heart fibrosis,keloids and hypertrophic scars.